Hepatitis A virus (HAV) is a picornavirus with a single-stranded RNA genome of approximately 7500 nucleotides. The wild-type strain of HAV grows poorly in cell-culture, generally is not cytopathic, and virus yields are low. A cell-culture adapted mutant has been selected which grows significantly more efficiently in cell-culture and which is attenuated for marmosets and chimpanzees. The objectives of this project are to determine the genetic basis for virulence and adaptation to cell culture of HAV in order to develop a strain of HAV suitable for use as an attenuated vaccine. The following advances in our understanding of HAV were made. In an effort to increase replicative capacity, chimeric viruses were constructed from two or more HAV strains including a virulent human strain, an attenuated strain, a vaccine strain, a cytopathic strain, and a simian strain. The P2 region from a cytopathic strain of HAV was shown to confer the large focus phenotype but not the lytic phenotype of the cytopathic virus. Both halves of the P2 region were involved but not all of the mutations were required. HAV/7 chimeras containing the 2C gene of the simian virus grew less efficiently than did HAV/7 but intragenic 2C chimeras grew at an intermediate level. The first phenotype linked to the 2A gene was identified when we found that an engineered point mutation in 2A caused accumulation of viral capsids in the nucleus. Chimeric viruses were constructed and assayed for the purpose of defining virulence genes. We identified the 2A gene as a second major HAV/7 determinant of attenuation for marmosets and obtained evidence that mutations in the 2A and 2C genes are almost totally responsible for the attenuation of the virus. The genetic determinants of virulence could be conferred through the 2C gene of a simian virus. Engineered mutations in the 2A gene decreased signs of histopathology but did not lower serum liver enzyme levels. Large deletions in the 5' noncoding region did not affect virulence but 5' noncoding mutations required for growth in MRC-5 cells resulted in lowered serum liver enzyme levels in combination with significant histopathology and high levels of virus excretion. A full length infectious cDNA of the attenuated HAV MRC-5 cell-adapted vaccine strain was constructed to serve as a genetic repository for the vaccine strain and to permit detailed molecular analysis of the virus it encodes.